The Role Of FAK In The Secretion Of MMP9 After CD147 Stimulation In Macrophages

Backgrounds and ObjectiveACS?acute coronary syndrome?is still one of the leading causes of mortality^[1,2],even if there has been great improvement in treatment.The formation of thrombus as the progression of vulnerable plaque is an important cause of acute coronary syndromes.The key features of vulnerable plaques include large necrotic core,thin fibrous cap and abundant foam cells^[3].Great advances have been made in the signaling pathways involved in the formation of unstable plaques for developing therapeutic methods.However,despite the considerable efforts,the mechanisms underlying the vulnerable plagues remain incompletely understood.The imbalance which leads to plaque rupture is between the extracellular matrix?ECM?deposition and matrix destruction.CD147,first identified in tumor cells and also known as extracellular MMP inducer?EMMPRIN?,can induce the production of various Matrix metalloproteinases?MMPs?^[4].Matrix metalloproteinases?MMPs?,a family of zinc-dependent proteinases capable of degrading various structural components of ECM,thus lead to ECM destruction and plague rupture^[5].These findings motivate further investigation of the mechanism of MMPs secretion in ACS.MMP9,secreted by macrophages,is used as an important marker for detecting vulnerable plagues^[6,7].Focal adhesion kinase?FAK?is a PTK?protein tyrosine kinase?,activated by cell attachment to the extracellular matrix and play a role in regulating cell attachment and motility^[8,9].Wu,Hao^[10]found MMP2 expression were elevated after CD147 stimulation in hepatocellular carcinoma,together with FAK and pFAK protein expression.Therefore,FAK might be involved in secretion of MMP9 in macrophages.To investigate whether FAK?focal adhesion kinase?can participate in the secretion of MMP9?matrix metalloproteinase 9?after CD147 stimulation in THP-1 induced macrophages,thus to explore the potential treatment perspectives for ACS.Methods1.Cell culture and differentiationTHP-1 cells were purchased from Kunming Institute of Zoology.To differentiate THP-1 cells into macrophages,THP-1 cells were plated in RPMI 1640 media?Hyclone,China?with 10%fetal bovine serum?Hyclone,China?in CO₂ at 37?.THP-1 cells?1*10⁶/ml?were induced to differentiate into macrophages using 5ng/ml phorbol12-myristate 13-acetate?PMA??Solarbio,China?for 48h.Differentiation of PMA treated cells was enhanced after the initial 48h stimulus by removing the PMA-containing media and then incubating the cells in fresh RPMI 1640 for 24h.To explore the peak of MMP9 and FAK after CD147?Abcam,USA?stimulation,the mRNA expression levels and protein expression levels were assessed at 0h,3h,6h,9h,12h.2.Cell Viability AssayThe macrophages were treated with FAK inhibitor 14?Abcam,USA?at 10?M for different time.Cell viability was monitored by the colorimetric water-soluble tetrazolium salt?CCK8?assay using a Cell Counting Kit-8?beyotime,China?according to the manufacturer's instructions.The optical density on 96-plate was analyzed with a microplate reader at 450nm to determine cell viability.3.Quantitative Real-time Polymerase Chain Reaction?qRT-PCR?Total RNA was extracted by EastepTM Total RNA Super Extraction Kit?promega,China?according to the manufacturer's protocol.Complementary DNA was reversetranscribed using PrimeScript?RT reagent Kit?Takara,Japan?.qRT-PCR was performed by SYBR Premix Ex Taq Kit?Takara,Japan?.GAPDH was used as endogenous control for the normalization of gene expression.The forward and reverse primers for FAK were5'-TGGTGCAATGGAGCGAGTATT-3',for MMP9 were 5'-TCGTCATCGTCGAA ATGGGC-3'and 5'-GGGACGCAGACATCGTCATC-3'and for GAPDH were 5'-TCGGAGTCAACGGATTTGGT-3 and 5'-TTGCCATGGGTGGAATCATA-3'.The PCR cycle conditions composed of an initial denaturation step at 95�C for 30sec,followed by 40 cycles at 95�C for 5 sec,60�C for 30 sec.Relative mRNA expression levels were assessed by the 2^-??Ct method in comparison with control group.4.Western blotting analysisCells were lysed in Cell lysis buffer for Western blot?20 mM Tris pH 7.5,150 mM NaCl,1%Triton X-100??Beyotime,China?containing complete Protease and phosphatase inhibitor cocktail.Protein concentration was determined by the Enhanced BCA Protein Assay Kit?Beyotime,China?and gels loaded with equal amounts of protein per lane.Electrophoretic separation was carried out on 7%polyacrylamide gels?Beyotime,China?,and subsequently transferred to PVDF membrane?Beyotime,China?.Membranes were blocked in 5%non-fat dry milk powder in TBST buffer for 1h.Then the membranes were incubated at 4?overnight with primary antibodies:rabbit anti-FAK?Y695?monoclonal antibody?1:1000??Abcam,USA?,rabbit anti-FAK?Y397?monoclonal antibody?1:1000??Abcam,USA?,rabbit anti-MMP9 monoclonal antibody?1:1000??Abcam,USA?,followd by incubation of secondary antibodies:HRP-Goat Anti-Rabbit IgG?H+L??1:2000??proteintech?.GAPDH?1:2500??Abcam,USA?served as the loading control.ECL detection system?Beyotime,China?was used to detect protein bands.Results?1?Relative mRNA expression of FAK and MMP9 were both significantly up-regulated?all P<0.05?after stimulation of CD147,FAK peaked at 9h?3.908�0.106vs.1,P<0.05?,while MMP9 peaked at 6h?2.522�0.062 vs.1,P<0.05?.?2?Relative protein expression of FAK,pFAK and MMP9 were all significantly increased after CD147stimulation?all P<0.05?,FAK?1.930�0.024 vs.1,P<0.05?and pFAK?1.737�0.021 vs.1,P<0.05?peaked at 9h,while MMP9 peaked at 6h?1.527�0.033 vs.1,P<0.05?.?3?CD147up-regulates FAK,pFAK and MMP9 mRNA and protein expressions in a dose-dependent manner.?4?FAK inhibitor 14 significantly reduced the relative protein expression level of pFAK?0.077�0.012 vs.1,P<0.05?and MMP9?0.133�0.012?at 9h after CD147stimulation.ConclusionThe results demonstrated that FAK Y397 phosphorylation was involved in the secretion of MMP9 after CD147 stimulation in macrophages and may play a role in the regulation of ACS.